by Newton PenkoffLidbeck
Fun Rating: 5/5
Difficulty Rating: 4/5
What is the general purpose?
Multiphoton microscopy is a type of fluorescent light microscope that uses 2 or more photons (tiny light particles) to image a sample. Multiphoton microscopes are especially common in neuroscience research to capture the structure and function of brain cells (neurons). Multiphoton microscopes also allow us to see deeper inside of animals or tissues that are tagged with a fluorescent (glowing) protein without having to slice the tissue into thinner pieces.
Why do we use it?
Multiphoton microscopes allow scientists to image deeper into their samples without needing to slice them into thinner sections. For example, multiphoton microscopes can be used to observe how neurons deep inside the brain connect to other cells farther away, which would be impossible to observe with different microscopy techniques. Also, multiphoton microscopes can capture an image extremely quickly, such as imaging neurons firing together. Since the laser light is visible only at a tiny spot, there is less tissue damage. This is especially important when imaging a live animal or tissue, where the laser can “burn out” your fluorescent protein, making the fluorescent light dim.
(Figure legend: A simplified example of how the sample is exposed to laser light (green) in 1-photon microscopy compared to multiphoton or 2-photon microscopy. In 1-photon microscopy, the entire sample on the slide is exposed to laser light. A single photon (blue) excites the fluorescent protein, which shows a fluorescent signal (grey) as it loses energy during emission (orange). In multiphoton microscopy, only a small dot on the sample is exposed to laser light. Two photons (pink) excite the fluorescent protein, which similarly shows a fluorescent signal (grey) as it loses energy during emission (orange). Figure created with Biorender.)
How does it work?
Fluorescent proteins need light energy to glow. Lasers provide fluorescent proteins with a “packet” of light energy in the form of tiny particles called photons. The process where the fluorescent protein gets energy from the photon is called “excitation.” As the protein loses energy from the photon, it gives off glowing fluorescent light in a process called “emission.” In 1-photon microscopes, such as a confocal microscope, a single photon is used to excite the protein. In multiphoton microscopes, 2 photons are used. This is why most multiphoton microscopes are known as “2-photon microscopes.”
In Dr. Arnaldo Carreira-Rosario’s lab at Duke University, I study brain development in tiny fruit fly embryos. Since fruit flies grow quickly, I can record a video as their brain develops and “turns on” when neurons learn to talk to each other by forming circuits. I can record both the shape and function of these neurons using different fluorescent proteins. Using a fluorescent protein on the cell membrane, I can label and observe how the tiny branching structures of neurons grow and change over time. Using another fluorescent protein, I can see how calcium moves in and out of the neuron’s cell body in a quick pulse. This process allows me to visualize the initiation and transmission of a neuronal signal by tracking the fluorescent pulse.
If I used a 1-photon microscope, the brain activity of my embryos would change, as they would be able to sense the laser light and respond to it. Using the 2-photon microscope in my lab, I can capture multiple hours of brain development without damaging the embryos, and their brain activity remains unchanged as they’re unable to sense the tiny spot of laser light while I image them.
